ABSTRACT Failure to properly coordinate DNA replication with repair and potentially mutagenic translesion DNA synthesis (TLS) contributes to mutations that underlie numerous human disease states, including cancers, as well as antibiotic resistance and adaptation of clinically significant microbial pathogens. We use Escherichia coli as a model to define fundamental mechanisms by which organisms mange the actions of their high fidelity replicative DNA polymerase(s) (Pol) with those of low fidelity TLS Pols involved in lesion bypass and mutagenesis. The process by which one Pol replaces another at a replication fork is referred to as `Pol switching'. Sliding clamp proteins (DnaN or ? in bacteria; PCNA in eukaryotes and archaea) play essential roles in these switches. The generally accepted `toolbelt' model for Pol switching postulates that two different Pols simultaneously bind separate hydrophobic clefts in the same ? clamp to sequentially access the DNA. In stark contrast with the toolbelt model, we recently determined that TLS Pols interact with each other and with the Pol III replicase, and while the mechanistic contributions of these interactions are currently unknown, we nevertheless demonstrated that Pol-Pol interactions are absolutely required for Pol switching in vitro and in vivo. We have also identified several novel Pol-? clamp interactions important for Pol function. In Aim 1, we will exploit our structural model of the Pol III-? clamp-Pol IV complex, as well as a wealth of biochemical, biophysical, single molecule and genetic approaches to define for the first time in molecular detail the specific contributions to switching of discrete Pol III-Pol IV and Pol IV-? clamp interactions. In Aim 2 we will exploit structural insights we have gained regarding the Pol II-? clamp complex to define in molecular detail how the ? clamp manages Pol II processivity, proofreading and Pol III-Pol II and Pol II-Pol IV switching. In Aim 3, we will use small angle X-ray scattering (SAXS), cryo-electron microscopy (cryo-EM), molecular modeling and a combination of biochemical and biophysical approaches to structurally define how the different Pols interact with each other and the ? clamp, and how DNA influences these interactions. In addition, we will determine the protein stoichiometry of the different Pol-? clamp and Pol-? clamp-Pol complexes using size exclusion chromatography coupled with multi angle light scattering (SEC-MALS), which Pols interact with each other, and whether or not these interactions are competitive. Taken together, results of these studies will provide unprecedented insight into the molecular mechanisms by which E. coli manages and coordinately regulates the actions of its different Pols during DNA replication, repair and TLS. Finally, our findings may identify critical steps in these higher order regulatory networks that generalize to other bacteria and can be targeted by chemotherapeutics to control replication and mutagenesis.